Hepatitis B is the most common chronic infectious disease in the world. The hepatitis B virus (HBV) demonstrates considerable genetic diversity with seven genomic groups (designated genotype A through G) having been identified to date (Norder, H., et al., (1993) J. Gen. Virol., 74:1341–1348; Stuyver, L. et.al., (2000) Journal of General Virology, 81:67–74). Each of these genotypes shows a characteristic geographical origin, and comprises several variant HBV genomes. Worldwide molecular diversity of HBV is based on the variability of the S-gene. The maximum genetic divergence of HBV genomes has been determined at 8% over the complete genome (Magnius, L. O. and Norder, H., (1995) Intervirology, 38:24–34).
Characterisation of HBV by genotype is fairly recent. Historically HBV was characterised on the basis of immunological reaction of the hepatitis B surface antigen (HBsAg) with sets of monoclonal antibodies. Isolates were described as “a,” indicating the common determinant for all different subtypes, followed by the subtype specific combinations: dw, dr, yw, or yr. The latter are mutually exclusive pairs of determinants, covering the HBsAg amino acids 122 (d=lys, y=arg) and 160 (w=lys, r=arg).
Currently available methods to diagnose HBV infection are immunoassay-based techniques that rely on serological markers such as HbsAg, HbeAg, anti-HBc IgM, or anti-HBe, anti-HBs, or anti-HBc IgGs. Immunoassay techniques are by nature non-quantitative and, in addition, require detection of more than one serological marker in order to determine whether an individual is currently infected or has been infected in the past.
An assay capable of directly detecting HBV nucleic acids in the serum or plasma of an infected subject, rather than the presence of serological markers, would provide a distinct advantage over immunoassay techniques. For example, detection of serum levels of HBV nucleic acids would provide a means for direct quantitation of the amount of HBV present in a sample. With the advent of anti-viral therapy for the treatment of HBV infection, such direct quantitation of HBV in the serum or plasma has become essential in order to monitor the progress of this therapy.
Methods of detecting HBV nucleic acids have been previously proposed. For example, International Patent Application No. PCT/US93/09233 and European Patent Application Nos. 0 593 789 A1 and 0 860 505 A1 describe polymerase chain reaction (PCR) based assays for genotyping and detecting HBV, and U.S. Pat. No. 5,736,316 describes a sandwich hybridization assay for the detection of HBV nucleic acids. However, there is currently no single method available for detection of all known genotypes of HBV; most available techniques are purposely based on differences between genotypes thus allowing the genotypes to be distinguished. A need, therefore, exists for a method that allows detection of HBV nucleic acids regardless of genotype. Such a method would have worldwide applicability as a diagnostic tool. In addition, since currently available immunoassay techniques are not capable of measuring serum levels of viral nucleic acids, a need exists for a method that allows quantitative determination of HBV viral nucleic acids in an infected subject.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention. Publications referred to throughout the specification are hereby incorporated by reference in their entireties in this application.